P53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. In greater than 50% of human tumors, the negative regulatory activities of p53 can be inactivated by either gene mutation or gene deletion. The p53 tumor suppressor gene products are either inactive or absent and this condition contributes to malignant transformation. However, in the remaining human tumors that contain normal p53 genes and express normal p53 gene products, their negative growth activity is rendered inactive by unknown mechanisms within cancer cells. We are testing the hypothesis that normal functioning of tumor suppressor gene products in some tumors may be circumvented or functionally inactivated by differential phosphorylation of p53 or changes in pathways impinging on the p53 pathway. Changes in p53 phosphorylation state can regulate its transcriptional activity, sometimes without detectable changes in p53 levels. We are studying how changes in p53 phosphorylation might affect its biological activity. We are developing methods to assess p53 phosphorylation status. Recombinant-p53 from baculovirus was used as a model to develop a mass spectrometry (MS) means for site-specific determination of phosphorylation. Okadaic acid treatment created a hyperphosphorylated species. Six phosphorylation sites and N-terminal acetylation were identified by MS. 2D PAGE was also used separate p53 phosphoisoforms and we found that each isoform was a mixture of phosphorylated species. These MS studies set the stage for further analytical work linking changes in p53 phosphorylation with biological events such as proliferation, apoptosis and growth arrest. Altered phosphorylation of p53-dependent genes might also be an effective method of downstream p53 inactivation to escape growth control in tumor development. Our collaborators at the University of Alabama have tested the hypothesis that many clinical gliomas contain wild type p53 protein that is inactivated postranslationally. After discovering a high level of nitric oxide synthetase activity in these brain tumors, they showed immunoreactive nitrosylated tyrosine residues on p53. Using the purified p53 that our laboratory supplied them as a surrogate target, they were able to show tyrosine nitrosylated residues on r-p53 using extracts from clinical gliomas. Further, they demonstrated that nitrosylated p53 bound poorly to DNA containing sequence specific binding sites suggesting posttranslational inactivation of p53. Our collective studies suggest that posttranslation inactivation by phosphorylation by key kinases modifying p53 or by other enzyme systems that alter the p53 molecule can provide alternative explanation of how tumors circumvent the normal function of p53 without mutation or deletion of the p53 gene. These studies suggest alternate target sites for clinical therapy for those patients that possess malignancies containing normal p53 genes. Ongoing studies involve differing responses of normal diploid fibroblasts and tumor cells to ionizing radiation and oxidative stress where normal and tumor cells each contain wild type p53. We are comparing cell cycle arrest patterns, p53 phosphorylation status, expression of p53 downstream target genes and relevant kinases that affect the p53 pathway. These studies reveal that p53 protein undergoes different post-translational changes according to the type of DNA damage exposure. Ongoing studies are directing our attention to the effects of cell phenotype on the in vivo activity of wild type p53. Our recent studies are a logical extension of our prior work in which targeted proteomic analysis will be used provide further insight on differing phosphorylation and protein-protein interactions of p53 in normal and tumor cells.